Composition and method for prevention, mitigation or treatment of an enteropathogenic bacterial infection

ABSTRACT

Bicyclic compounds for decreasing the expression of bacterial virulence factors thereby preventing, mitigating, or treating bacterial infection are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/837,139 filed Aug. 27, 2015, which is a continuation-in-partapplication of U.S. patent application Ser. No. 13/384,860 filed Feb.23, 2012, which is a 35 U.S.C. § 371 filing of International ApplicationNo. PCT/US2010/042704 filed Jul. 21, 2010, which claims the benefit ofpriority to U.S. Provisional Application Ser. No. 61/301,264 filed Feb.4, 2010 and U.S. Provisional Application Ser. No. 61/227,190 filed Jul.21, 2009, the contents of which are incorporated herein by reference intheir entireties.

GOVERNMENT SUPPORT

This invention was made with government support under contract numbersAI072661 and AI039654 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

The increasing resistance of bacterial pathogens to antibiotics,combined with fundamental advances in understanding the mechanisms andregulation of bacterial virulence, has prompted the identification ofpathogen anti-virulence drugs that antagonize the activity of virulencefactors. Cholera is an acute intestinal infection caused by thebacterium Vibrio cholerae, a gram-negative flagellated bacillus. Inaddition to being a class B bioterrorism threat, cholera is morewidespread today than it was in the previous century. The expression ofV. cholerae's primary virulence factors, the toxin-coregulated pilus(TCP) and cholera toxin (CT), occurs via a transcriptional cascadeinvolving several activator proteins, and serves as a paradigm for theregulation of bacterial virulence. Strains of V. cholerae capable ofcausing the significant epidemics and pandemics of cholera that haveoccurred throughout history possess two genetic elements, the Vibriopathogenicity island (VPI) and the lysogenic CTX phage. Both of theseelements have inserted into the circular chromosome I and are present inthe pathogenic forms of the organism. The VPI contains the genesresponsible for the synthesis and assembly of the essential colonizationfactor TCP, and the CTX phage encodes the CT genes. Expression of theTCP and CT genes is coordinately regulated at the transcriptional levelby a virulence cascade involving activator proteins encoded both withinthe VPI and the ancestral genome. AphA and AphB initiate the expressionof the cascade by a novel interaction at the tcpPH promoter. AphA is amember of a new regulator family and AphB is a LysR-type activator, oneof the largest transcriptional regulatory families. Once expressed,cooperation between TcpP/TcpH and the homologous transmembraneactivators ToxR/ToxS activates the toxT promoter. ToxT, an AraC/XylS(A/X) type regulator, then directly activates the promoters of theprimary virulence factors. Thus, ToxT is the paramount regulator ofvirulence gene expression.

ToxT inhibitors have been identified and shown to provide protectionagainst intestinal colonization by V. cholerae. For example, bile(Schuhmacher, et al. (1999) J. Bacteriol. 181:1508-14) and several ofits unsaturated fatty acid constituents, i.e., oleic acid, linoleicacid, and arachidonic acid (Chatterjee, et al. (2007) Infect. Immun.75:1946-53) have been shown to inhibit virulence factor gene expression.Similarly, virstatin, a small molecule4-[N-(1,8-naphthalimide)]-n-butyric acid, has been shown to inhibitvirulence regulation in V. cholerae (Hung, et al. (2005) Science310(5748):670-4). Further, U.S. Pat. No. 5,866,150 teaches compoundshaving the structure: CH₃(CH₂)_(n)—CH═CH—CH₂CH═CH—(CH₂)—R—COOR′ for usein treating bacterial infections including, e.g., S. aureus, V. cholera,S. dysenteria, B. substilis, and S. typhemurium.

High resolution structure of ToxT has shown that ToxT contains an almostcompletely buried and solvent inaccessible sixteen carbon fatty acidbound to a pocket in the N-terminal domain, which can influence its DNAbinding activity. In particular, virulence gene expression can bereduced between 6-8 fold with cis-palmitoleic acid and 10-15 fold witholeic acid (Lowden, et al. (2010) Proc. Natl. Acad. Sci. USA107:2860-5).

SUMMARY OF THE INVENTION

This invention provides a compound having the structure of Formula I, orhydrates, isomers, prodrugs or pharmaceutically acceptable salts ofFormula I:

wherein R¹ is hydrogen or polar group; R² is hydrogen, alkenyl, or oxo;X is —CHCH—, —NH—, —C═N—, —N═C—, —C—NH— or —NH—C—; n is 0-6; and dashedlines represent bonds that are independently present or absent. Apharmaceutical, nutraceutical, nutritional, medical nutrition food orfunctional food composition containing a compound of Formula I is alsoprovided.

This invention also includes methods for decreasing expression of abacterial virulence factor and preventing, mitigating, or treating aninfection by a bacterium that expresses an A/X regulatory protein usinga compound of Formula I. In some embodiments the pathogenic bacterium isVibrio cholerae, Escherichia coli, Shigella flexneri, Yersiniaenterocolitica, Yersinia pestis, Brucella abortus Salmonella typhi,Bacillus anthracis, Clostridium botulinum, Listeria monocytogenes,Staphylococcus aureus or Salmonella typhimurium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence alignment of A/X family members from V. cholerae(Vc ToxT; SEQ ID NO:1), an E. coli ETEC strain (Ec FapR; SEQ ID NO:2),an E. coli EPEC strain (Ec PerA; SEQ ID NO:3), S. typhi (St SirC; SEQ IDNO:4), S. typhimurium (St HilD; SEQ ID NO:5), S. flexneri (Sf VirF; SEQID NO:6), Y. enterocolitica (Ye VirF; SEQ ID NO:7), an E. coli ETECstrain (Ec Rns; SEQ ID NO:8), Y. pestis (Yp AraC; SEQ ID NO:9), B.anthracis (Ban AraC; SEQ ID NO:10), B. abortus (Bab AraC; SEQ ID NO:11)and C. botulinum (Cb AraC; SEQ ID NO:12). Secondary structurepredictions and homology modeling indicate common helix (H), loop/turn(L) and lysines or other positive amino acids (underlined residues) atpositions homologous to those identified in ToxT. The entire sequencelengths were compared, but only partial sequences are shown here.

FIG. 2 shows the effects of compound 1 on tcp expression. V. choleraecells were grown in LB pH 6.5 at 30° C. for 18 hours±the indicatedinhibitor in methanol. β-galactosidase activity of a tcp-lacZ reporterconstruct was determined. β-galactosidase units are shown for awild-type strain treated with methanol (MeOH), 0.01% oleic acid (0.01%OA), 0.02% oleic acid (0.02% OA), or 0.02% compound 1 (Comp1) ascompared to an untreated control (ToxT) and an untreated ToxT deletionstrain (ΔToxT).

DETAILED DESCRIPTION OF THE INVENTION

A class of bicyclic compounds has now been identified, which exhibitsanti-virulence activity against V. cholerae. The compounds of thisinvention were designed to bind to the pocket located in the N-terminusof ToxT (Lowden, et al. (2010) Proc. Natl. Acad. Sci. USA 107:2860-5),thereby disrupting DNA binding activity and virulence gene expression.Given that ToxT homologues are found in a wide variety bacterialpathogens, the compounds of this invention find use as broad spectrumanti-virulence agents in the treatment of antibiotic-resistant bacterialinfections as well as in prophylactic treatment of infections, e.g., fortravelers or military personnel in areas with suboptimal water and/orfood quality. Moreover, because the compounds of this invention arehighly specific for pathogens, the normal bacterial flora of the gut isnot affected.

Compounds of this invention have the structure of Formula I, whichincludes hydrates, isomers, prodrugs or pharmaceutically acceptablesalts of Formula I:

wherein R¹ is hydrogen (H) or polar group, e.g., a C₁-C₆ alkyl, C₁-C₆alkenyl, C₄-C₆ aryl, halo (e.g., F, Cl, or Br) CF₃, OCH₃, NO₂, CN, OH,NMe₂, COOH, or COOCH₃; R² is hydrogen (H), alkenyl (═CH₂), or oxo (═O);X is —CHCH—, —NH—, —C═N—, —N═C—, —C—NH— or —NH—C—; n is 0-6; and dashedlines represent bonds that are independently present or absent. Incertain embodiments, n is 3, 4, or 5.

In some embodiments, the invention is a compound having the structure ofFormula II, which includes hydrates, isomers, prodrugs orpharmaceutically acceptable salts of Formula II:

wherein R¹ is a polar group, e.g., a C₁-C₆ alkyl, a C₁-C₆ alkenyl, C₄-C₆aryl, halo, CF₃, OCH₃, NO₂, CN, OH, NMe₂, COOH, or COOCH₃; X is —CH—,—NH—, —C═N—, —N═C—, —C—NH— or —NH—C—; R² is hydrogen (H), alkenyl(═CH₂), or oxo (═O); and dashed lines represent bonds that areindependently present or absent.

As used herein, the term “alkyl” refers to a straight or branched chainhydrocarbon, preferably having from one to six carbon atoms (i.e.,C₁-C₆). Examples of “alkyl” as used herein include methyl, ethyl,propyl, isopropyl, n-butyl, isopentyl, n-pentyl, and the like, as wellas substituted versions thereof. In particular embodiments, an alkyl ofthe invention is a C₁-C₃ alkyl.

The term “alkenyl” refers to a straight or branched chain aliphatichydrocarbon containing one or more carbon-to-carbon double bonds thatmay be optionally substituted, with multiple degrees of substitutionincluded within the present invention. Examples include, but are notlimited to, vinyl, allyl, and the like, as well as substituted versionsthereof.

The term “aryl” refers to a monovalent group with an aromatic carbonatom as the point of attachment, said carbon atom forming part of afour-, five- or six-membered aromatic ring structure wherein the ringatoms are all carbon, and wherein the monovalent group is composed ofcarbon and hydrogen. Non-limiting examples of aryl groups includephenyl, methylphenyl, (dimethyl)phenyl, -ethylphenyl, propylphenyl,—C₆H₄CH(CH₃)₂, —C₆H₄CH(CH₂)₂, methylethylphenyl, vinylphenyl, naphthyl,and the monovalent group derived from biphenyl. In particularembodiments, the aryl is a phenyl group.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

Exemplary compounds of the invention include, but are not limited to:

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, and whichpossess the desired pharmacological activity. Such salts include acidaddition salts formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or with organic acids such as 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid,3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylicacid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and di-carboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

Compounds of the invention may also exist in prodrug form. Sinceprodrugs are known to enhance numerous desirable qualities ofpharmaceuticals, e.g., solubility, bioavailability, manufacturing, etc.,the compounds employed in some methods of the invention may, if desired,be delivered in prodrug form. Thus, the invention contemplates prodrugsof compounds of the present invention as well as methods of deliveringprodrugs. Prodrugs of the compounds employed in the invention may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compound. Accordingly, prodrugs include, forexample, compounds described herein in which a hydroxy or carboxy groupis bonded to any group that, when the prodrug is administered to apatient, cleaves to form a hydroxy or carboxylic acid, respectively. Forexample, a compound comprising a hydroxy group may be administered as anester that is converted by hydrolysis in vivo to the hydroxy compound.Suitable esters that may be converted in vivo into hydroxy compoundsinclude acetates, citrates, lactates, phosphates, tartrates, malonates,oxalates, salicylates, propionates, succinates, fumarates, maleates,methylene-bis-β-hydroxynaphthoate, gentisates, isethionates,di-p-toluoyltartrates, methanesulfonates, ethanesulfonates,benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates,esters of amino acids, and the like.

Compounds of the invention can be prepared as described herein (seeExample 1) or using any suitable methodology routinely practiced in theart, and be analyzed for their pharmacological properties by routinemethodologies. For example, kinetic solubility can be measured using adirect UV absorbance method or thermodynamic solubility can be measured.In addition, stability in gastrointestinal fluids can be determined byconventional methods (Asafu-Adjaye, et al. (2007) J. Pharm. Biomed.Anal. 43:1854-1859), e.g., 1 hour in simulated gastric fluid (pH 1.2,pepsin) at 37° C. and/or 3 hours in simulated intestinal fluid (pH 6.8,pancreatin). Furthermore, using the Parallel Artificial MembranePermeability Assay (PAMPA)-blood-brain barrier (BBB) permeability assay(Di, et al. (2009) J. Pharm. Sci. 98:1980-1991) or B-P dialysis (Kalvass& Maurer (2002) Biopharm. Drug Dispos. 23(8):327-38), brain penetrationcan be assessed. Furthermore, lipophilicity can be estimated bypartitioning between octanol and water using a shake flask method or pHmetric method and permeability can be assessed using the Caco-2 celllayer method of PAMPA assay.

A compound of this invention may be administered in a pharmaceuticalcomposition by various routes including, but not limited to,intradermal, intramuscular, intraperitoneal (e.g., by injection),intravenous, subcutaneous, intranasal, epidural, oral, sublingual,intracerebral, transdermal, rectal, or topical administration. Dependingon the route of administration, the active compound may be coated. Forexample, to administer the therapeutic compound by a route other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation. By way of illustration, the therapeutic compound may beadministered to a subject in an appropriate carrier, for example,liposomes, or a diluent. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan, et al. (1984) J. Neuroimmunol. 7:27).

When the compound is to be administered parenterally, intraperitoneally,intraspinally, or intracerebrally, dispersions can be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compound can be orally administered, for example, with an inertdiluent or an assimilable edible carrier. The therapeutic compound andother ingredients may also be enclosed in a hard or soft shell gelatincapsule, compressed into tablets, or incorporated directly into thesubject's diet. For oral therapeutic administration, the therapeuticcompound may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a subject.

In addition to conventional pharmaceutical compositions, this inventionalso provides nutraceuticals; nutritional compositions, such as dietarysupplements; medical nutrition or functional foods including a compoundof Formula I. Such compositions can be prepared by mixing one or morecompounds of the invention with an edible nutritionally acceptable solidor liquid carriers and/or excipients, e.g., fillers, such as cellulose,lactose, sucrose, mannitol, sorbitol, and calcium phosphates; andbinders, such as starch, gelatin, tragacanth, methylcellulose and/orpolyvinylpyrrolidone (PVP). Optional additives include lubricants andflow conditioners, e.g., silicic acid, silicon dioxide, talc, stearicacid, magnesium/calcium stearates and polyethylene glycol (PEG)diluents; disintegrating agents, e.g., starch, carboxymethyl starch,cross-linked PVP, agar, alginic acid and alginates, coloring agents,flavoring agents and melting agents. The composition of the inventioncan optionally include conventional food additives, such as emulsifiers,stabilizers, sweeteners, preservatives, chelating agents, osmoticagents, buffers or agents for pH adjustment, acidulants, thickeners,texturizers and the like.

In addition to the above, the compositions of the present invention canfurther include antibiotics (e.g., tetracyclines), probiotics,prebiotics, anti-LPS sIgA (Apter, et al. (1993) Infect. Immun.61(12):5279-5285), as well as other monounsaturated fatty acids such asoleic acid or palmitoleic acid to facilitate the prevention, mitigationand/or treatment of a bacterial infection. As such, compositionscontaining other monounsaturated fatty acids such as oleic acid,palmitoleic acid and vaccenic acid and their use in the treatment ofbacterial infections are also embraced by the present invention.

Suitable product formulations according to the present invention includesachets, soft gel, powders, syrups, pills, capsules, tablets, liquiddrops, sublinguals, patches, suppositories, liquids, injectables and thelike. Also contemplated are food and beverage products containing one ormore compounds of the present invention, such as solid food products,like bars (e.g., nutritional bars or cereal bars), powdered drinks,dairy products, breakfast cereals, muesli, candies, confectioneries,cookies, biscuits, crackers, chocolate, chewing-gum, desserts and thelike; liquid comestibles, like soft drinks, juice, sports drinks, milkdrinks, milk-shakes, yogurt drinks or soups, etc. The addition of one ormore compounds of the invention to animal feed is also included withinthe scope of this invention.

The compositions of the invention can be provided as a component of ameal, e.g., a nutritional or dietary supplement, in the form of a healthdrink, a snack or a nutritionally fortified beverage, as well as aconventional pharmaceutical, e.g., a pill, a tablet or a softgel, forexample.

ToxT belongs to the AraC/XylS (A/X) superfamily of regulatory proteins.This family is composed of approximately 1,974 members identified in 149bacterial genomes including Bacillus anthracis, Listeria monocytogenesiand Staphylococcus aureus (Ibarra, et al. (2008) Genetica 133:65-76),and is known for its role in virulence gene regulation. The crystalstructure of ToxT identified a binding pocket enclosed by residues Y12,Y20, F22, L25, I27, K31, F33, L61, F69, L71, V81, and V83 from theN-terminal domain and residues I226, K230, M259, V261, Y266, and M269from the C-terminal domain. The volume of this predominantly hydrophobicpocket is 780.9 Å³ as calculated by the program CASTp. This pocketcontains a sixteen-carbon fatty acid with a negatively chargedcarboxylate head group forming salt bridges with both K31 from theN-terminal domain and K230 from the C-terminal domain. Using secondarystructure prediction and homology modeling, multiple candidates from theA/X protein superfamily were identified, which contained lysines orother positive amino acids at positions homologous to those identifiedin ToxT. This analysis indicated that A/X regulatory proteins from manypathogenic bacteria, including Vibrio cholerae, Escherichia coli,Shigella flexneri, Yersinia enterocolitica, Yersinia pestis, Brucellaabortus, Salmonella typhi, Bacillus anthracis, and Clostridium botulinum(FIG. 1), as well as Staphylococcus aureus and Salmonella typhimuriumcontain with homologous lysine residues and/or homologous ligand bindingpockets. Thus, use of compositions herein can be broadly applied totreat enteric bacterial infections that cause travelers' diarrhea,salmonella, brucellosis, botulism, dysentery, and typhoid fever,diseases infecting some 4 billion people annually worldwide.

Thus, the present invention embraces compositions containing one or morecompounds of the invention for use in methods for decreasing orinhibiting the expression of bacterial virulence genes. This method iscarried out by contacting a pathogenic bacterium with a composition ofthe present invention so that the expression of at least one virulencefactor, e.g., TCP and/or CT in V. cholerae, is measurably decreased ascompared to bacteria not contacted with the composition of theinvention. A decrease or inhibition of virulence factor expression canbe measured using any conventional method for monitoring nucleic acid orprotein levels in a cell, e.g., northern blot analysis, RT-PCR analysis,dot blot analysis, western blot analysis and the like. Desirably, thecomposition of the invention decreases virulence factor expression by10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or as much as 100% ascompared to untreated bacteria.

V. cholerae. There are several characteristics of pathogenic V. choleraethat are important determinants of the colonization process. Theseinclude adhesins, neuraminidase, motility, chemotaxis and toxinproduction. If the bacteria are able to survive the gastric secretionsand low pH of the stomach, they are well adapted to survival in thesmall intestine. V. cholerae is resistant to bile salts and canpenetrate the mucus layer of the small intestine, possibly aided bysecretion of neuraminidase and proteases. Specific adherence of V.cholerae to the intestinal mucosa is likely mediated by the longfilamentous TCP pili which are coregulated with expression of thecholera toxin genes.

As indicated herein, V. cholerae produces cholera toxin, which iscomposed of two A subunits and five B subunits. The B subunits allowbinding to a ganglioside (GM₁) receptor on the intestinal epithelialcells. The B pentamer must bind to five corresponding GM₁ receptors.This binding occurs on lipid rafts, which anchor the toxin to themembrane for endocytosis of the A subunits, thereby trafficking thetoxin into the cell and to the basolateral surface where it acts (Lencer(2001) Am. J. Physiol. Gastrointest. Liver Physiol. 280:G781-G786). Onceinternalized, the A subunits proteolytically cleave into A1 and A2peptides. The A1 peptide ADP-ribosylates a GTP-binding protein, therebypreventing its inactivation. The always active G protein causesadenylate cyclase to continue forming cAMP. This increase inintracellular cAMP blocks absorption of sodium and chloride bymicrovilli and promotes the secretion of water from the intestinal cryptcells to preserve osmotic balance (Torgersen, et al. (2001) J. Cell Sci.114:3737-3747). This water secretion causes the watery diarrhea withelectrolyte concentrations isotonic to plasma. The fluid loss occurs inthe duodenum and upper jejunum, with the ileum less affected. The colonis less sensitive to the toxin, and is therefore still able to absorbsome fluid. The large volume, however, overwhelms the colon's absorptivecapacity.

In addition to V. cholerae, the following is a list of some of thebacterial enteric pathogens that express A/X family members thatproperly align with ToxT. In so far as other pathogens may be identifiedbased upon the structural analysis disclosed herein, the following listis merely illustrative and in no way limits the scope of bacteria thatcan be targeted by the instant fatty acid compositions.

Escherichia coli. There are several pathogenic derivatives of E. coli.Several of the most common are as follows. One is Enterohemorrhagic E.coli (EHEC), which causes a Shigella-like illness and is also known asthe hamburger meat E. coli. Another is Enteropathogenic E. coli (EPEC),which causes persistent diarrhea in children. EPEC expresses a surfaceappendage termed the bundle forming pilus, or BFP. BFP is required forintestinal colonization by the bacterium. BFP gene expression isactivated by the A/X family member PerA that meets alignment criteriadescribed herein. A third example is Enterotoxigenic E. coli (ETEC),which expresses a toxin identical to ToxT and causes traveler'sdiarrhea. ETEC expresses colonization factor adhesions termed CS1 andCS2. The expression of the corresponding genes is activated by an A/Xfamily regulator termed Rns that meets alignment criteria describedherein. Similarly, the cof gene cluster, Longus gene cluster and CFA/Ioperon of ETEC also respectively encode regulatory proteins cofS, lngSand CfaD, which regulate the expression of virulence factors. Indeed,CfaD and Rns are fully interchangeable with each other (Bodero, et al.(2007) J. Bacteriol. 189:1627-32) and recognize the same DNA bindingsites.

Salmonella. Salmonella cause 1.4 million cases of gastroenteritis andenteric fever per year in the US and lead all other food borne pathogensas a cause of death. While there are over a thousand serotypes ofSalmonella that can cause gastroenteritis, S. enteritidis (sv.Typhimurium) is the leading cause. S. enteritidis (sv. Typhimurium)infection of mice serves as a model for typhoid fever as the causativeagent of this disease only infects humans. As such, this species hasserved as a model organism for both gastroenteritis and typhoid fever.Most of the genes that encode virulence factors are located in clusterson salmonella pathogenicity islands termed SPIs. SPI-1 carries the genesfor a type III secretion system (T3SS), the expression of which iscritical for virulence. The master regulator of the expression of SPI-1genes is HilA. The expression of HilA itself is controlled by HilD. HilDis an A/X family member that meets alignment criteria described herein.

Salmonella typhi (S. enterica sv. Typhi) is the leading cause of entericfever also known as typhoid fever. Typhoid fever is estimated to affectapproximately 17 million people annually, causing 600,000 deaths. S.typhi is a multi-organ organism, infecting lymphatic tissues, liver,spleen, and bloodstream. S. typhi has a gene regulatory network similarto the SPI-1 and regulation of T3SS gene expression in S. enteritidis(sv. Typhimurium). In the case of S. typhi the aligned A/X family memberis designated SirC.

Shigella. Several Shigella species are responsible for the majority ofbacillary dysentery that is caused by this organism. S. dysenteriae iscommon in many parts of the world. S. flexneri and S. sonnei are themost common in the U.S. Most molecular analysis regarding Shigella hasbeen performed with S. flexneri. This species requires a surfaceprotein, IcsA, to nucleate actin and travel through and between hostcells. Expression of the icsA gene is activated by VirF, which meetsalignment criteria described herein.

Bacillus anthracis. Bacillus anthracis is an aerobic spore-formingbacteria that causes anthrax disease. Livestock may become infected byeating or inhaling anthrax spores. Humans, especially farmers andindividuals who work in slaughterhouses, may develop cutaneous anthraxthrough skin exposure to infected animals. Humans can also getinhalational anthrax by breathing in material contaminated with thebacteria. This bacterium also expresses an AraC family member.

Listeria. Listeria monocytogenes is a facultative intracellularbacterium that is the causative agent of Listeriosis. It is one of themost virulent food-borne pathogens with 20 to 30 percent of clinicalinfections resulting in death. Listeria monocytogenes also expresses anAraC family member.

Staphylococcus aureus. Staphylococcus aureus is a facultativelyanaerobic, gram-positive coccus and is the most common cause of staphinfections. Some strains of S. aureus, which produce the exotoxinTSST-1, are the causative agents of toxic shock syndrome, whereas otherstrains of S. aureus also produce an enterotoxin that is the causativeagent of S. aureus gastroenteritis.

Yersinia enterocolitica is a common pathogen of children and adults,with a strong propensity for extraintestinal complications.Gastrointestinal disorders include enterocolitis, particularly inchildren, and pseudoappendicitis, particularly in young adults. Y.enterocolitica virulence factors include outer proteins termed Yops andYadA, which is an adhesin that is essential for colonization. VirF is anA/X family member that meets alignment criteria described herein.

Yersinia pestis is the pathogen of human and animals that causes allthree main forms of the plague including pneumonic, septicemic andbubonic plagues. Similar to VirF, the Y. pestis virulence regulon iscontrolled by the A/X family member LcrF (Hoe, et al. (1992) J.Bacteriol. 174:4275-86).

In addition, Bacillus anthracia, the etiologic agent of anthrax;Brucella abortus, which causes brucellosis; and Clostridium botulinum,the causal agent of botulism, each have an A/X family member that meetsalignment criteria described herein.

In so far as ToxT and other A/X regulatory proteins directly regulatethe expression of virulence factors, which are involved inpathogenicity, inhibition of A/X regulatory protein activity, and hencevirulence factor expression, is useful in the prevention, mitigation,and/or treatment of enteropathogenic bacterial infection. As usedherein, the term “bacterial infection” is used to describe the processof adherence and virulence factor production by a pathogenic bacteriumthat expresses an A/X regulatory protein. For the purposes of thepresent invention, the term “treatment” or “treating” means anytherapeutic intervention in a mammal, preferably a human or any otheranimal suffering from a enteropathogenic bacterial infection, such thatsymptoms and bacterial numbers are reduced or eliminated. By way ofillustration, it is contemplated that by reducing adhesion of V.cholerae to the intestinal mucosa via TCP pili, colonization will bereduced or inhibited, thereby allowing the subject to clear thebacterial infection.

“Prevention” or “preventing” refers to prophylactic treatment, whereinclinical symptom development is delayed or inhibited, e.g., preventinginfection from occurring and/or developing to a harmful state.

“Mitigation” or “mitigating” means arresting the development of clinicalsymptoms, e.g., stopping an ongoing infection to the degree that it isno longer harmful, or providing relief or regression of clinicalsymptoms, e.g., a decrease in fluid loss resulting from an infection.

Prophylactic or therapeutic treatment involves the administration of aneffective amount of a compound of this invention to a subject in needthereof, thereby preventing, mitigating, or treating a bacterialinfection. Subjects benefiting from the method of the invention includethose having a bacterial infection (e.g., exhibiting signs or symptoms)or those at risk of having a bacterial infection (e.g., a subjectexposed to a contaminated food or water source).

The terms “effective amount” means a dosage sufficient to measurablydecrease or inhibit virulence gene expression and provide prevention,mitigation and/or treatment of a bacterial infection. In prophylactic ortherapeutic applications, preferably the administered dose delays,mitigates, or reduces the signs and/or symptoms of infection in thesubject by at least about 20%, more preferably by at least about 40%,even more preferably by at least about 60%, and still more preferably byat least about 80% relative to untreated subjects. The efficacy of acompound can be evaluated in an animal model system that may bepredictive of efficacy in preventing, mitigating or treating the diseasein humans.

The amount and dosage regimen of the composition of the invention to beadministered is determined in the light of various relevant factorsincluding the purpose of administration (e.g., prevention, mitigation ortreatment), the age, sex and body weight of an individual subject,and/or the severity of the subject's symptoms. In this respect, thecompositions of the invention can be administered under the supervisionof a medical specialist, or may be self-administered.

Daily dosage of a composition of the present invention would usually besingle or multiple servings per day, e.g., once or twice daily, foracute or chronic use. However, benefit may be derived from dosingregimens that can include consumption on a daily, weekly or monthlybasis or any combination thereof. Administration of compositions of theinvention, e.g., treatment, could continue over a period of days, weeks,months or years, until an infection has been treated. Optimally, thecomposition of the invention is consumed at least once a day on aregular basis, to prevent an infection.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1: Design and Synthesis of Compounds

Based upon the structure of palmitoleic acid, small molecule inhibitorsof ToxT were designed. A synthetic method for preparing exemplarycompounds of the invention is presented in Scheme 1.

Compound 1 was tested in a reporter assay to determine its effect oninhibiting the activity of ToxT. This analysis indicated that compound 1effectively inhibited the activity of ToxT as determined in a tcpreporter assay (FIG. 2).

In light of the activity of compound 1, this invention also includesderivatives of compound 1. To modify activity, specificity, and/orbioavailability, additional derivatives of compounds 1-3 can beprepared. For example, by varying the anhydride (or a functionalizeddicarboxylic acid), the carbon chain linking the naphthalene ring to thecarboxylate head group can be lengthened or shortened. Thus, reaction of1-bromo-8-methylnaphthalene with glutaric anhydride will afford acompound similar to compounds 1-3 having three carbons between theketone carbonyl and the carboxylate group. Likewise, carrying out theacylation reaction with pimelic acid anhydride will give a compoundsimilar to compounds 1-3 having five carbons between the ketone carbonyland the carboxylate group. Accordingly, the acylation reaction can becarried out with any suitable dicarboxylic acid, e.g., malonic,succinic, glutaric, adipic, pimelic or suberic acid/anhydride, toachieve a compound having between 1 and 6 carbons between the ketonecarbonyl and the carboxylate group.

Further, the methyl group attached to the naphthalene ring can besubstituted with any other alkyl or polar group, either by de novosynthesis from a suitable 2-alkylfuran and 3-bromobenzyne (see synthesisof 1-bromo-8-methylnaphthalene in Scheme 1) or by modification of themethyl group in the final product. For example, appropriate oxidationwill convert this methyl group to a carboxylate or to a hydroxyl group.Of note, the synthesis of 1-bromo-8-methylnaphthalene automaticallygives the isomeric 1-bromo-5-methylnaphthalene, which provides a set ofcontrol compounds isomeric to compounds 1-3.

The aromatic carbonyl ketone group can be readily reduced with sodiumborohydride in trifluoroacetic acid (Gribble, et al. (1978) Synthesis763) or other two-step reduction procedures known in the art. Theresulting compounds can be further reduced by catalytic hydrogenation toa derivative of compound 3. By adjusting the reduction conditions (e.g.,Birch reduction), compounds having one reduced ring (i.e., a tetralinanalogue [1,2,3,4-tetrahydronaphthalene]) can be prepared.

As a further derivative, the ketone carbonyl in Compound 1 can beconverted to an alkene, so as to “stiffen” the carbon chain.

In certain embodiments, the naphthalene ring is replace with4-methylindole, which could be deemed an isostere to8-methylnaphthalene. Introduction of carboxylate chains onto the indoleC-3 position is facile as this position is extremely susceptible toelectrophilic substitution. Moreover, the indole double bond is readilyreduced, providing additional flexibility for polarity of the basictwo-ring structure. This is shown in Compound 4.

Similarly, the 8-methylnaphthalene unit can be replaced with theisostere 5-methylquinoline. Once again, the C-4 position of quinoline,like C-3 in indole, is very easily substituted and will provide a ringstructure for conversion to derivatives of compound 1 and, by reduction,to derivatives of compound 3. This is shown in compound 5, and, forisoquinoline, compound 6.

What is claimed is:
 1. A compound or a hydrate, isomer, orpharmaceutically acceptable salt thereof having the structure:

wherein R¹ is C₂-C₆ alkyl; R² is hydrogen, alkenyl, or oxo; X is —CHCH—,—NH—, —C═N—, or —N═C—; when X is —CHCH—, R² is hydrogen, alkenyl, oroxo; when X is —NH—, —C═N—, or —N═C—, R² is hydrogen, or oxo; n is 1-6.2. The compound of claim 1, wherein X is —NH—, —C═N—, —N═C—.
 3. Thecompound of claim 1, wherein X is —CHCH—.
 4. The compound of claim 1,wherein n is 3, 4, or
 5. 5. The compound of claim 1, wherein n is 1, 2,3, 4, or
 5. 6. The compound of claim 1 having the structure:


7. The compound of claim 1 having the structure:


8. A pharmaceutical composition comprising the compound of claim 1 inadmixture with a pharmaceutically acceptable carrier.
 9. A compoundselected from the group consisting of:

and hydrates, isomers, or pharmaceutically acceptable salts thereof. 10.A pharmaceutical composition comprising the compound of claim 9 inadmixture with a carrier.
 11. The compound of claim 1 having thestructure:


12. The compound of claim 1 having the structure:

wherein n is 1.